The products of the cocoa bean, i.e. cocoa, chocolate, and cocoa butter, originally developed by the Aztecs and adopted by the Spanish, are more prized today than ever,
Unfortunately, the cocoa tree Theobroma cacao grows only in tropical regions 10.degree.-15.degree. N & S of the equator. The productivity of the cocoa tree is limited and expansion in production is slow. Further, political changes are affecting the reliability of supply, as well as the quality of the product.
General information on the cocoa bean and chocolate can be found in the books: Cooke, L. R., "Chocolate Production and Use", Magazine for Industry, New York (1963) and Minifie, B. W., "Chocolate, Cocoa and Confectionary: Science and Technology", Churchill, London (1970).
Cocoa butter is the premium confectionary fat because of its special physical properties, its unique taste and flavor. There is an increasing demand for cocoa butter and though some reasonable imitations or extenders are available, their use is limited by regulatory as well as technical problems. When cocoa butter is mixed with these imitation butters they form mixtures with inferior softening and melting characteristics as compared to pure cocoa butter.
While there are numerous publications on the unique composition and structure of cocoa butter triglycerides and their economically important physical properties, there is a dearth of information concerning the biosynthesis of cocoa bean lipids.
The ripe bean has one of the highest lipid contents (55-60 percent fat) of any occurring natural product. This is preponderantly 98+% triglyceride with 1-2% polar lipids [Parsons et al., Am. Oil Chem. Soc., 46:425 (1969)].
The fatty acid composition of cocoa butter has been reported. Palmitic acid, (C16), stearic acid (C18), oleic acid (C18:1) and linoleic acid (C18:2) account for 26, 35, 34 and 3 percent of the total fatty acids respectively; [Jurriens et al, J. Am. Oil Chem. Soc., 47:344 (1970); Kavanagh et al, J. Am. Oil Chem. Soc., 42:9 (1965); Iverson, J. Assoc. Off. Anal. Chem., 55:1319 (1972)].
The triglyceride composition of cocoa butter has been reported by many investigators; [Vander Val, J. Amer. Oil Chem. Soc., 37:18 (1960); Coleman, J. Amer. Oil Chem. Soc., 38:685 (1961); Subbaram et al, J. Am. Oil Chem. Soc., 41:445 (1964); Chacko et al, J. Am. Oil Chem. Soc., 41:843 (1964); Jurriens et al, (1965), supra]. There is general agreement on the triglyceride composition of cocoa butter. The stereospecific distribution of the fatty acids in cocoa butter triglycides (99% of total lipid) was reported by several workers; [Jurriens et al, (1965) supra; Youngs, J.. Am. Oil Chem. Soc., 38:62 (1961); Feuge et al, J. Am. Oil Chem. Soc., 50:50 (1973)]. Oleic acid and linoleic acid are located exclusively on Sn-2 (.beta.) whereas palmitic acid and stearic acid are equally distributed on SN-1 and SN-3 (.alpha.,.alpha.').
Of most significance, however, is the marked non-random distribution of these fatty acids in the glycerides. Cocoa butter is unique because it is preponderantly composed of three triglyceride species--2-oleodipalmitin, POP (15%); 2-oleodistearin, SOS (25%), and 2-oleopalmitostearin, POS (40%). Thus 80% of the triglycerides are symmetrical (SUS type), and it is these that account mostly for the desirable physical properties of cocoa butter.
The hardness (fatty acid composition) of cocoa butter varies with mean daily temperature during growth; Alvin et al Revista Theobroma, 2:3 (1972). Winter crop cocoa developing in regions with a lower mean temperature produced cocoa butter of higher iodine value (softer). This was confirmed by Lehrian [Ph. D. Dissertation, Penn State University, (1978)] who studied changes in composition and melting characteristics of cocoa butter in relation to the state of maturity of cocoa beans and temperatures. It was reported that cocoa lipids progressively accumulated in maturing cocoa seeds from 120 days through 160 days after pollination. The butter from pods grown at higher temperatures had slightly more palmitate and stearate and 3% less oleate (harder). This phenomenon is consistent with the observation of other plants in tissue culture showing that lower growth temperatures increases the biosynthesis of unsaturated fatty acids; [Radwan et al, Adv. in Lipid Res., 14:171, Academic Press (1976)].
There is abundant information concerning the synthesis of lipids in certain plants (soy, avocado, corn, safflower, pea) and this has recently been reviewed in comprehensive volumes by Hitchcock "Plant Lipid Biochemistry", Academic Press, N.Y. (1971) and Galliard and Mercer "Recent Advances in the Chemistry and Biochemistry of Plant Lipids", Academic Press, N.Y. (1975). Noteworthy is the absence of any information on the biosynthesis of cocoa butter.
Limited research has been done on exploiting plant cell cultures for their capacity to produce useful metabolities. Thus, certain cells in culture have been shown to synthesize aromatics and essential oils, Camborg, Expl. Cell Res., 50:151 (1968).
Until recently, plant cell suspension cultures has not been employed extensively for studies related to lipid metabolism. The literature on this subject was reviewed by Radwan et al., Adv. in Lipid Res., 14:171, Academic Press (1976). Moore et al., Ph. Physiol., 53:261 (1974) have described a number of parameters involving the use of soybean suspension cells. Stearns et al., Phytochem., 14:619 (1975), have examined the capacity of soybean suspension cells to incorporate acetate into fatty acids as well as the effect of growth hormones on lipid biosynthesis. Stumpf et al., Lipids 12:120 (1977), demonstrated that soybean cell suspension actively absorbed exogenous (C16 and C18) fatty acids desaturated them and converted them into triglycerides typical of soybean oil. The limited number of plant tissues examined in culture did not accumulate triglycerides under the conditions used, though Jones, Industrial Aspects of Biochem., Amsterdam, P. 813 (1974), has reported that an increased synthesis of triglycerides occur with the induction of embryogenesis in cultures of rape tissue.
The techniques used for all culture are adapted from microbiological techniques and entail growing sterile tissue or cells in sterile nutrient media. Gamborg et al., "Paint Tissue Culture Methods" Nat'l Res. Comm., Saskatoon, Canada (1975), have recently described the experimental techniques used in plant tissue culture. The plant tissue to be cultured is sterilized, usually with sodium hypochlorite solution, and after washing is placed in sterile culture medium under aseptic conditions. The medium contains all of the known nutrients required for plant cells. The requirements vary with different plant species but generally sucrose or glucose, inorganic nitrogen (salts), some amino acids, the major and minor (trace) mineral elements, vitamins, and some auxins are included. The appropriate medium has to be determined for each plant species but some universal media are now available. In some instances media in which other cells have been grown contain stimulatory compounds which aid the establishment of primary cultures [Street et al., "Les cultures de Tissues de Plants", Colloq. N193 p. 17, CNRS, Paris (1971)].